1. Field of the Invention
The present invention relates generally to the fields of organic synthesis of bioactive compounds. More particularly, it concerns methods of synthesis for N-homocysteine thiolactonyl retinamido cobalamin ("thioretinaco"), a compound that has potential antineoplastic, anticarcinogenic, antiatherogenic, chemopreventive, antiviral, and antiaging activities.
N-homocysteine thiolactonyl retinamido ("thioretinamide") is a starter material for the preparation of thioretinaco.
2. Description of Related Art
McCully (U.S. Pat. No. 4,618,685; "the '685 patent", incorporated herein by reference) attempted to synthesize thioretinamide, the precursor to the compound of the invention, from homocysteine thiolactone free base and retinoic acid. McCully's synthesis of thioretinamide involves the conjugation of homocysteine thiolactone to retinoic acid using dicyclohexylcarbodimide as a coupling agent. The process in that patent starts with the preparation of the free base of homocysteine thiolactone. This is done by dissolving sodium hydroxide (NaOH) in water, adding methylene chloride and rapidly mixing. Homocysteine thiolactone hydrochloride is then slowly added to the mixture. After 15 minutes of mixing, the methylene chloride layer is separated, dried over anhydrous sodium sulfate and the solvent is evaporated under reduced pressure at 37.degree. C. The resulting clear liquid (homocysteine thiolactone free base) is immediately added to tetrahydrofuran (or any other non-polar solvent) containing all-trans-retinoic acid. Then, dicyclohexylcarbodiimide is added, and the reaction mixture is stirred 16 hours at 20.degree. C., protected from light. The tetrahydrofuran is removed at 37.degree. C. under reduced pressure, and the yellow-white residue is added to 500 ml of water and 500 ml of ethyl acetate. The mixture is stirred vigorously for one hour, and the ethyl acetate layer is separated and dried over anhydrous sodium sulfate. The ethyl acetate is concentrated to about 20 ml at 50.degree. C. under reduced pressure and cooled. The resulting N-homocysteine thiolactonyl retinamide, (a yellow powder) was produced with a 69% theoretical yield and a melting point of 172.degree. C.
Unfortunately, it has recently been shown that the method described in the '685 patent does not produce thioretinamide in pure form. The inventors of the present invention attempted the method of the '685 patent, as described in McCully and Vezeridis, 1987a, and were unsuccessful in obtaining sufficient, pure thioretinamide as confirmed by subsequent analysis by 300 MHz N.M.R. Proton N.M.R. analysis of the compounds produced by McCully's method did not give conclusive evidence that the compound produced was thioretinamide. When the present inventors attempted to repeat the method described in the '685 patent, they conclusively showed that a major product of this procedure was the byproduct dicyclohexylurea (DCU). In sum, the procedure described in the above-listed references did not produce thioretinamide in the expected quantity or purity. Therefore, this procedure did not provide an efficient basis for producing thioretinamide for use in the preparation of thioretinaco.
Despite the difficulties in synthesizing thioretinamide following the procedures described in the '685 patent, the work of McCully showed that there was promise for thioretinamide as a therapeutic. The "thioretinamide" synthesized as described was able to counteract the carcinogenecity of ethyl carbamate in pulmonary neoplasms of strain A female mice. Thioretinamide decreased the number of tumors formed to 80% at doses of 50 mg/week, and 60% at doses of 200 mg/week. Unfortunately, the mice receiving the higher dose showed significant weight loss (McCully and Vezeridis, 1987a).
In U.S. Pat. No. 4,925,931 ("the '931 patent"; incorporated herein by reference), thioretinamide has been shown to react with cobalamin to form N-homocysteine thiolactonyl retinamido cobalamin, also known as thioretinaco. Both thioretinamide and thioretinaco have anticarcinogenic and antineoplastic activities. (McCully and Vezeridis, 1987a; McCully and Vezeridis, 1989). In experiments with cultured malignant and normal cells, thioretinaco was found to have antiproliferative activity, and thioco, the complex of homocysteine thiolactone and cobalamin, was found to increase growth of both malignant and normal cells. (McCully et al., 1992). Intratumoral administration of thioretinaco decreased the growth of human pancreatic adenocarcinomas in athymic mice. (McCully et al., 1989).
The above-described studies and patents pointed to great promise for thioretinamide and thioretinaco as chemotherapeutic agents. However, in order to fully realize the potential of this agent, methods of producing them in sufficient quantities and purity are needed. Co-pending U.S. Pat. No. 6,054,595, filed Jun. 29, 1999, and issued Apr. 25, 2000, incorporated herein by reference in its entirety, describes the development of methods that produces high yields of pure N-homocysteine thiolactonyl retinamide (thioretinamide).